Synthetic phosphoethanolamine-modified oligosaccharides reveal the importance of glycan length and substitution in biofilm-inspired assemblies

Bacterial biofilm matrices are nanocomposites of proteins and polysaccharides with remarkable mechanical properties. Efforts understanding and tuning the protein component have been extensive, whereas the polysaccharide part remained mostly overlooked. The discovery of phosphoethanolamine (pEtN) modified cellulose in E. coli biofilms revealed that polysaccharide functionalization alters the biofilm properties. To date, the pattern of pEtN cellulose and its mode of interactions with proteins remains elusive. Herein, we report a model system based on synthetic epitomes to explore the role of pEtN in biofilm-inspired assemblies. Nine pEtN-modified oligosaccharides were synthesized with full control over the length, degree and pattern of pEtN substitution. The oligomers were co-assembled with a representative peptide, triggering the formation of fibers in a length dependent manner. We discovered that the pEtN pattern modulates the adhesion of biofilm-inspired matrices, while the peptide component controls its stiffness. Unnatural oligosaccharides tune or disrupt the assembly morphology, revealing interesting targets for polysaccharide engineering to develop tunable bio-inspired materials.


Supplementary Notes
All chemicals used were reagent grade and used as supplied unless otherwise noted. The automated syntheses were performed on a home-built synthesizer developed at the Max Planck Institute of Colloids and Interfaces. Analytical thin-layer chromatography (TLC) was performed on Merck silica gel 60 F254 plates (0.25 mm). Compounds were visualized by UV irradiation or dipping the plate in a staining solution (sugar stain: 10% H2SO4 in EtOH; CAM: 48 g/L ammonium molybdate, 60 g/L ceric ammonium molybdate in 6% H2SO4 aqueous solution). Flash column chromatography was carried out by using forced flow of the indicated solvent on Fluka Kieselgel 60 M (0.04 -0.063 mm). Analysis and purification by normal and reverse phase HPLC was performed by using an Agilent 1200 series. Products were lyophilized using a Christ Alpha 2-4 LD plus freeze dryer. 1 H, 13 C and HSQC NMR spectra were recorded on a Varian 400-MR (400 MHz), a Varian 600-MR (600 MHz) or a Varian 700-MR (700 MHz) spectrometer. Spectra were recorded in CDCl3 by using the solvent residual peak chemical shift as the internal standard (CDCl3: 7.26 ppm 1 H, 77.0 ppm 13 C) or in D2O using the solvent as the internal standard in 1 H NMR (D2O: 4.79 ppm 1 H). High resolution mass spectra were obtained using a 6210 ESI-TOF mass spectrometer (Agilent) and a MALDI-TOF autoflex TM (Bruker). MALDI and ESI mass spectra were run on IonSpec Ultima instruments. Scanning electron microscopy (SEM) images were obtained with a Gemini SEM, LEO 1550 system with cold field emission gun operation at 3 kV. All the samples were coated with Au/Pd. Transmission electron microscopy (TEM) images were obtained on carbon-coated copper grids with a Zeiss EM 912Ω instrument at 120 kV without negative staining. Circular dichroism (CD) spectra were acquired with a Chrascan qCD spectrometer (Applied Photophysics Ltd. Leatherhead, UK) using a quartz cuvette (Helma GmbH & Co. KG, Mullheim, Germany) at 23°C with a band width of 1 nm. The thioflavin T (ThT) fluorescence was measured at RT using a SpectraMax M5 plate reader (Molecular Devices LLC., California, USA) with an excitation wavelength at 438 nm with a cut-off filter at 475 nm. The 5 day-matured samples were incubated with the ThT solution with the final concentration of 20 µM for 30 minutes and stirred 10 seconds right before the measurement. Atomic force microscopy (AFM) was carried out in air with a JPK NanoWizard 4 AFM. Images were attained with the conventional AC mode and flattened without further modification. The samples for AFM for the imaging of single filaments (Day 1) and matured fibrils (Day 5) were prepared on freshly cleaved mica. Qualitative imaging (QI) mode was applied for nanoindentation and adhesion force measurement with a silicon cantilever. For mechanical property analysis, the uniform film with the thickness of 300 nm were prepared on a pre-washed glass substrate. The nanoindentation was conducted in air with a silicon nitride cantilever at a constant force of 10 nN. The approaching force-distance curve were fit to the Hertz model and manipulated to obtain Young's modulus. A tipless cantilever was modified with a polystyrene bead (diameter, 8.4 µm) for the adhesion measurement. 10 x 10 curves were obtained from one spot. A minimum of three samples with 10 spots per sample was tested for each oligosaccharide. JPK data processing software was used to analyze all AFM data including images and forces. All solutionstate NMR experiments regarding R5 and its interaction with oligosaccharides were performed using a Bruker Ascend TM (AvanceIII HD) 700 MHz NMR spectrometer with water suppression. All spectra were recorded at 297 K. In order to monitor the aggregation of R5, 1 H spectra were acquired on Day 0, Day 1, and Day 5. Data were processed with MestReNova. Automated glycan assembly (AGA)

General materials and methods
The automated syntheses were performed on a home-built synthesizer designed at the Max Planck Institute of Colloids and Interfaces. All solvents used were HPLC-grade. The solvents used for the building block, activator, TMSOTf and capping solutions were taken from an anhydrous solvent system (Jcmeyersolvent systems). The building blocks were co-evaporated three times with toluene and dried for 1 h under high vacuum before use. Activator, capping, deprotection, acidic wash and building block solutions were freshly prepared and kept under argon during the automation run. All yields of products obtained by AGA were calculated on the basis of resin loading. Resin loading was determined following previously established procedures. 5

Preparation of stock solutions
 Building block solution: 0.08 mmol of building block was dissolved in DCM (1 mL).  Activator solution: 1.35 g of recrystallized NIS was dissolved in 40 mL of a 2:1 mixture of anhydrous DCM and anhydrous dioxane. Then triflic acid (55 μL) was added. The solution is kept at 0 °C for the duration of the automation run.

Modules for automated synthesis
Module A: Resin Preparation for Synthesis (20 min) All automated syntheses were performed on 0.0125 mmol scale. Resin was placed in the reaction vessel and swollen in DCM for 20 min at RT prior to synthesis. During this time, all reagent lines needed for the synthesis were washed and primed. After swelling, the resin was washed with the DMF, THF, and DCM (three times each with 2 mL for 25 s).

Module B: Acidic Wash with TMSOTf Solution (20 min)
The resin was swollen in 2 mL DCM and the temperature of the reaction vessel was adjusted to -20 °C. Upon reaching the desired temperature, TMSOTf solution (1 mL) was added dropwise to the reaction vessel. After bubbling for 3 min, the acidic solution was drained and the resin was washed with 2 mL DCM for 25 s.

Module C: Thioglycoside Glycosylation (35 min)
The building block solution (0.08 mmol of BB in 1 mL of DCM per glycosylation) was delivered to the reaction vessel. After the set temperature was reached, the reaction was started by drop wise addition of the activator solution (1.0 mL, excess). The glycosylation conditions are building block dependent 1,6 (we report the most common set of conditions). After completion of the reaction, the solution was drained and the resin was washed with DCM, DCM:dioxane (1:2, 3 mL for 20 s) and DCM (two times, each with 2 mL for 25 s). The temperature of the reaction vessel was increased to 25 °C for the next module.
Supplementary The resin was washed with DMF (two times with 2 mL for 25 s) and the temperature of the reaction vessel was adjusted to 25 °C. 2 mL of pyridine solution (10% in DMF) was delivered into the reaction vessel. After 1 min, the solution was drained and the resin washed with DCM (three times with 3 mL for 25 s). 4 mL of capping solution was delivered into the reaction vessel. After 20 min, the solution was drained and the resin washed with DCM (three times with 3 mL for 25 s). Module E1: Fmoc Deprotection (9 min) The resin was washed with DMF (three times with 2 mL for 25 s) and the temperature of the reaction vessel was adjusted to 25 °C. 2 mL of Fmoc deprotection solution was delivered to the reaction vessel and kept under Ar bubbling. After 5 min, the solution was drained and the resin washed with DMF (three times with 3 mL for 25 s) and DCM (five times each with 2 mL for 25 s). The temperature of the reaction vessel was decreased to -20 °C for the next module.

Supplementary
Supplementary 4 was prepared according to a previously established procedure. 8 The partially protected oligosaccharide obtained from Module F was mixed with 4 (4 equiv.), coevaporated with pyridine for three times and dried under high vacuum for 2 h. The ratio between the oligosaccharide and 4 changed depending on the oligosaccharide structure (here we report the most common set of conditions, variations are reported in the specific procedures). The mixture was dissolved in anhydrous pyridine (2 mL) and a solution of pivaloyl chloride (equimolar to 4) in pyridine (1 mL) was added. The solution was stirred for 12 h at RT, after which time iodine (10 equiv.) and water (0.5 mL) were added and the reaction was stirred for additional 2 h. The reaction mixture was quenched with Na2S2O3 and extracted with CH2Cl2. The organic layers were combined and evaporated.

Module I: Hydrogenolysis at ambient pressure a
The crude compound obtained from Module H was dissolved in 2 mL of t-BuOH:H2O (1:1). The Pd catalyst (2.5 times the weight of the starting material) was added and the reaction was stirred in a flask equipped with a H2 balloon. The reaction progress was monitored to avoid undesired side products formation. Upon completion, the reaction was filtered and washed with t-BuOH and H2O. The filtrates were concentrated in vacuo.
a Reaction times and type of catalyst are indicated for each synthesis.

Module J: Purification
After photovleavage, crudes were analyzed and purified using analytical and preparative HPLC (Agilent 1200 Series spectrometer, Method A1 and Method A2, respectively). The protected phosphorylated crudes were purified with Method B and Method C. After methanolysis, the semi-deprotected compounds were purified with Method C. The final compounds were purified with Method E and analyzed using analytical HPLC (Agilent 1200 Series spectrometer, Method F). Following final purification, all deprotected products were lyophilized on a Christ Alpha 2-4 LD plus freeze dryer prior to characterization. Step
Step Modules Notes  Step Modules Notes Compound A2P2A2 was obtained as a white solid (1.7 mg, 9 % overall yield).
Step Modules Notes The solid-phase peptides synthesis was carried out with a microwave-assisted peptide synthesizer (Liberty Blue, CEM, USA). 2-Cl-Trt-Cl Protide resin was swollen in dichloromethane for 30 min. The first amino acid was coupled manually using 4 equiv. of Fmoc-Tyr(OtBu)-OH (with respect to the resin loading) and 8 equiv. of diisopropylethylamine (DIEA) in 3 mL of DCM, shaking at RT overnight.

Assembly of artificial fibers
Stock solutions were prepared dissolving separately R5 and the oligosaccharides in HFIP with a concentration of 200 µM (0.4 mg mL -1 ) and 0.13 mg mL -1 , respectively. The R5 and oligosaccharide stock solutions were mixed with 2 to 1 (or 1 to 1) volume ratio to reach the final mass ratio with 6 to 1 (or 3 to 1) and sonicated for 10 minutes. HFIP was removed under gentle nitrogen purging followed by evaporation under high vacuum. Complete HFIP removal was confirmed by 19 F-NMR. Water was added to the dried films to reach the final peptide concentration of 25 µM for imaging, CD, ThT binding test, and AFM force measurement, and 200 µM for 2D TOCSY NMR analysis. AFM imaging and force measurement were performed in air in an AFM chamber with a relative humidity (RH) of 25%. If not mentioned, the standard ratio between R5 and oligosaccharide is 6 to 1 by mass.
Supplementary Figure 49. Cartoon representation of the sample preparation method.

Fibrils structural analysis
Supplementary Figure 50. CD spectra of R5 in HFIP (black) adopting an alpha-helix structure and in water (red) adopting a beta-sheet structure (25 µM, 23 °C).
Supplementary Figure 51. CD spectra of R5 in PBS buffer (1X, pH 7.4) confirming that R5 showed the same secondary structure in water (neutral pH) and PBS buffer. Due to the strong background signal of the PBS buffer solution from 190 to 200 nm, the spectra were obtained from 200 to 250 nm. To avoid artefacts originated from buffer solutions and obtain clear images, all the samples in the following experiments were prepared in water.
Supplementary Figure 53. Time-dependent conformational changes of R5 in the presence of different hexasaccharides monitored by CD at λ194 nm (black) and λ215 nm (red). While the structural transition of R5 alone reached a plateau within 20 minutes, longer times are required in the presence of the hexasaccharides.
Supplementary Figure 60. ThT emission spectra to test the presence of amyloid fibers in the R5 samples incubated for 5 days with/without different hexasaccharides, showing maxima at around 482 nm 9 . Intensity (a.u.) Supplementary Figure 64. 1 H NMR of R5 recorded at different time intervals. Broadening of the signals and decreased intensity indicate aggregation followed by precipitation due to the formation of aggregates.
NMR comparison of the four samples.
Supplementary Figure 71. Overlay of the 1 H-1 H TOCSY NMR recorded for R5 alone (gray) and the samples in the presence of A6 (blue), (PA)3 (red) and P2APA2 (yellow). For clarity, only the region of the amidic proton is shown.